Shwu-Huey Liaw

Crystal structure of glucooligosaccharide oxidase from Acremonium strictum: a novel flavinylation of 6-S-cysteinyl, 8alpha-N1-histidyl FAD

Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-Å resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8α-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8α-methyl group of the isoalloxazine ring with Cys130 and His70, respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a β-d-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin N5. Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.

Structural Characterization of Glucooligosaccharide Oxidase from Acremonium strictum

ABSTRACT Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.

Functional Roles of the 6-S-Cysteinyl, 8α-N1-Histidyl FAD in Glucooligosaccharide Oxidase from Acremonium strictum

The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8α-positions of the flavin isoalloxazine ring cross-linked to Cys130 and His70, respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of ∼+69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with kcat values reduced to ∼2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.

Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

ABSTRACT Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the kcat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

HLA‐DRB1*0701 and DRB1*1401 are associated with genetic susceptibility to psoriasis vulgaris in a Taiwanese population

We analysed the allelic frequencies of class II human leucocyte antigen (HLA)‐DRB1, DQA1, DQB1 and DPB1 by polymerase chain reaction/sequence‐specific oligonucleotide probe hybridization typing in 76 Taiwanese psoriasis vulgaris (PSV) patients and 238 Taiwanese non‐psoriatic controls. The analysis revealed the following: (i) the DRB1*0701 allele was positively associated with PSV (relative risk, RR = 6.4, corrected P‐value, Pc ≤ 0.001); (ii) the DRB1*1401 allele was positively associated with type I PSV (age at onset < 40 years) (RR = 3.5, Pc ≤ 0.001); (iii) the DQA1* 0501 allele was negatively associated with PSV (RR = 0.4, Pc ≤ 0.001); (iv) there was no significant association of HLA‐DP genes with PSV; and (v) there was a strong association of β‐chain phenylalanine at position 37 (Phe 37) and glutamate or glutamine at position 74 (Glu 74/Gln 74) with PSV (RR = 3.5, Pc ≤ 0.001 for the association of Phe 37 with PSV; RR = 2.2, Pc ≤ 0.001 for the association of Glu 74/Gln 74 with PSV). The positive association between PSV and the DRB1*0701 allele is consistent with previous reports. The negative association of the DQA1* 0501 allele is reported only in Finland, whereas the positive association between PSV and the DRB1*1401 allele has never been described before. Trans‐racial studies may shed further light on the association of class II HLA alleles or other closely linked genes with the development of PSV. Phe 37 (a large, non‐polar amino acid) and Glu 74/Gln 74 (both negatively charged amino acids) were the polymorphic residues in pockets 9 and 4, respectively, of the β‐chain, which may have increased their affinity for the small non‐polar amino acids and basic amino acids of the psoriatic antigen peptide, thereby activating the T lymphocytes. This finding may facilitate the identification of a psoriatic antigen.

Crystal Structure of a Bifunctional Deaminase and Reductase from Bacillus subtilis Involved in Riboflavin Biosynthesis

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-Å resolution displayed a tetrameric ring-like structure with an extensive interface of ∼2400 Å2/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

ABSTRACT The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.

HLA-Cw6 specificity and polymorphic residues are associated with susceptibility among Chinese psoriatics in Taiwan.

Abstract Previous serotyping in Chinese patients has failed to confirm an association between HLA-Cw6 and psoriasis. As serotyping has proved to be less valuable in the determination of HLA-C, genotyping of HLA-C in 68 Taiwanese psoriasis vulgaris (PSV) patients was performed using polymerase chain reaction/sequence-specific oligonucleotide probe hybridization (PCR-SSOPH) of HLA-Cw genes. Compared to 213 non-PSV control subjects, HLA-Cw6 was significantly associated with PSV (16.18% of PSV patients vs 5.16% of controls; odds ratio 3.53, Pc≤10–4). The amino acid residues on the heavy chain of the HLA-Cw antigens from exon one to exon five were also analyzed. Asp 9, Ser 24, Ala 73, Asp 90, Trp 97, Trp 156 and Asn 77/Lys 80 were found to be positively associated with PSV (Pc≤0.0044, 0.0008, 0.0026, 0.0014, 0.039, 0.0020 and 0.0000, respectively).

Stable single-mode operation of an 850-nm VCSEL with a higher order mode absorber formed by shallow Zn diffusion

We report that an 850-nm vertical-cavity surface-emitting laser with a planar higher order mode absorber, formed by shallow Zn diffusion (<0.3 /spl mu/m), operated at stable single-mode over the entire drive current range. A device with a 5/spl times/5 /spl mu/m/sup 2/ absorber aperture and a 5/spl times/5 /spl mu/m/sup 2/ oxide confined active region showed a /spl sim/0.8 mA threshold and a mode suppression ratio of 40 dB. The modeling indicates that the higher order modes will be suppressed strongly due to the much larger threshold gain, compared to that of the fundamental mode as long as the Zn diffusion depth outside the 5/spl times/5 /spl mu/m/sup 2/ absorber aperture is over /spl sim/0.2 /spl mu/m, which agrees well with the experimental results.

Structural‐based mutational analysis of d‐aminoacylase from Alcaligenes faecalis DA1

d‐Aminoacylase is an attractive candidate for commercial production of d‐amino acids through its catalysis in the zinc‐assistant hydrolysis of N‐acyl‐d‐amino acids. We report here the cloning, expression, and structural‐based mutation of the d‐aminoacylase from Alcaligenes faecalis DA1. A 1,007‐bp PCR product amplified with degenerate primers, was used to isolate a 4‐kb genomic fragment, encoding a 484‐residue d‐aminoacylase. The enzyme amino‐terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D‐PSSM to be similar to urease and dihydroorotase, which have grouped into a novel α/β‐barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His‐67, His‐69, and His‐250, putative metal ligands in d‐aminoacylase, have been mutated previously, the remaining histidine (His‐220) and aspartate (Asp‐366) Asp‐65, and four cysteines were then characterized. Substitution of Asp‐65, Cys‐96, His‐220, and Asp‐366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc‐binding ability, revealing that Cys‐96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino‐terminal segment including Asp‐65 may be responsible for structural stabilization.